Airway inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are characterized by mucous cell metaplasia and mucus overproduction. This application addresses a central issue in the pathogenesis of these diseases by investigating a novel mechanism responsible for the overproduction of mucins during airway inflammation. Inflammation of human bronchial epithelia (HBE) activates the unfolded protein response (UPR). The UPR activation couples to stimulation of the inositol requiring enzyme 1 (IRE1), an ER enzyme that activates the transcription factor X-box binding protein 1 (XBP-1), the canonical IRE1-dependent pathway required for protein secretion in many cells. Although recent studies have shown that activation of the IRE1/XBP-1 pathway is functionally relevant for airway epithelial inflammation-mediated cytokine secretion in vitro and in vivo, the role of IRE1 signaling in mucin production during airway inflammation has not been investigated. Because IRE1 can also activate JNK and p38 MAP kinases and NF-?B (non-canonical IRE1 pathways), and their activation can induce mucin transcription, IRE1 might also be functionally relevant for mucin production. IRE1 exists in two isoforms, 1 and 2. We found that IRE1a was ubiquitously expressed, but IRE1[unreadable] was present only in gut and respiratory epithelia. While IRE1[unreadable] expression was >30 fold higher than IRE1a expression in HBE, IRE1[unreadable] was absent in primary human alveolar type II cells. Moreover, a strong correlation between IRE1[unreadable]2 and MUC5B expression was found during HBE differentiation. In mouse tissues, IRE1[unreadable] was absent in lung parenchyma but its expression was 6.5-8.5 fold higher than IRE1a expression in nasopharynx, trachea, and bronchus, tissues that exhibit higher levels of mucous cells. In wild-type (wt) mice, IRE1[unreadable] was expressed in Clara cells and ovalbumin (OVA)-induced mucous cells, but was absent in ciliated cells, suggesting a specific function for IRE1[unreadable] related to mucin production. OVA-increased airway epithelial Muc5b and Muc16 production was blunted in IRE1[unreadable]-/- vs. wt mice. Further evidence for a key functional role of IRE1[unreadable] in mucin production was given by a strong correlation between IRE1[unreadable] and genes involved in mucin production or glycosylation. Our Specific Aims will test the primary hypothesis that IRE1[unreadable] is required for mucin production by airway mucous cells, it stimulates mucin transcription and/or regulates genes involved in mucin production or glycosylation, its action is distinct from IRE1a action, and knocking out IRE1[unreadable] will reduce mucin overproduction in models relevant for human airways inflammatory disease. Studies will utilize in vitro and in vivo models of airway inflammation characterized by mucous cell metaplasia and mucin overproduction. Primary cultures of wt and IRE1[unreadable]-/- murine airway epithelia, primary cultures of HBE expressing different levels of IRE1[unreadable], and the Scnn1b mouse exhibiting deletion of IRE1[unreadable] in airway epithelia will be used to test the role of IRE1[unreadable] in mucin production. These novel studies may have a high impact by revealing IRE1[unreadable] as a therapeutic target for asthma, COPD, and CF airways disease. PUBLIC HEALTH RELEVANCE: Mucin overproduction characterizes many airway inflammatory diseases. Our studies will test the functional role of IRE1[unreadable], a protein involved in endoplasmic reticulum stress responses, in mucin production associated with airway inflammation and may lead to new therapies for asthma, COPD and CF patients.